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First published online 11 March 2003
doi: 10.1242/jcs.00385

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HIF-1 controls extracellular matrix synthesis by epiphyseal chondrocytes

David Pfander^1,2,*, Thorsten Cramer^2,*, Ernestina Schipani³ and Randall S. Johnson^2,

¹ Division of Orthopedic Rheumatology, Department of Orthopedic Surgery, University of Erlangen-Nuremberg, 91054 Erlangen, Germany
² Molecular Biology Section, Division of Biology, University of California San Diego, La Jolla, CA 92093, USA
³ Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA

View larger version (100K): [in a new window]   Fig. 1. Growth and morphology of wild-type and HIF-1-null epiphyseal chondrocytes. (A) The growth curves of wild-type chondrocytes compared with cells with Cre-recombinase-induced deletion of HIF-1. The increase of HIF-1-null chondrocytes (exponential phase) is strongly retarded compared with wild-type cells. All data points are representing means ± standard deviations of duplicates from two independent experiments. All cells were infected with MOI 100 of adenovirus containing either Cre recombinase or ß-galactosidase. In the lower panel, wild-type chondrocytes (+/+) on days 3 (B) and 5 (D) post-plating are compared with mutant chondrocytes lacking HIF-1 (-/-) (C,E). For the first few days in mutant cultures, single chondrocytes showed a bipolar or stretched cell shape. At day 5 post-plating, chondrocytes from both cultures were mostly confluent and appeared round or polygonal (D,E).

View larger version (22K): [in a new window]   Fig. 2. Free ATP and lactate levels of wild-type and mutant chondrocytes cultured under normoxia and 22 hours hypoxia. (A) Bars representing means ± standard deviations of free ATP-levels measured in three independent experiments, normalized to protein content. Free ATP levels were determined in mutant and wild-type cultures under normoxia and hypoxia. (B) Lactate levels in supernatants of wild-type chondrocytes cultured at 0.5% oxygen were significantly increased compared with normoxic levels. Reduced lactate concentrations were detected in supernatants of HIF-1-null chondrocytes under normoxia and hypoxia. Bars are representing the means ± standard deviations of measured triplicates from three independent experiments, normalized to protein concentrations. Statistical differences were calculated as: wild-type normoxia vs HIF-1-null normoxia; wild-type hypoxia vs HIF-1-null hypoxia; wild-type normoxia vs wild-type hypoxia. *P0.05; **P0.01.

View larger version (17K): [in a new window]   Fig. 3. Quantification of phosphoglycerate kinase-1, glucose transporter-1 and VEGF mRNA levels in wild-type and HIF-1-null chondrocytes. Chondrocytes were exposed to either 0.5% or 20% oxygen for 22 hours. (A) Bars represent the severalfold changes in PGK-1 mRNA levels compared with wild-type chondrocytes under normoxia (onefold expression). (B) Bars represent the severalfold changes in Glut-1 mRNA levels compared with wild-type chondrocytes under normoxia (normoxic expression taken as 1.0). (C) Bars represent the severalfold changes in VEGF mRNA levels compared with wild-type chondrocytes under normoxia (onefold expression). Data are given as means ± standard deviations (triplicates from three independent experiments). Statistical differences were calculated as: wild-type normoxia vs HIF-1-null normoxia; wild-type hypoxia vs HIF-1-null hypoxia; wild-type normoxia vs wild-type hypoxia. *P0.05; **P0.01.

View larger version (13K): [in a new window]   Fig. 4. Quantification of soluble VEGF isoforms. Soluble VEGF isoforms (VEGF 120 and VEGF 164) were determined in conditioned medium after either 22 hours hypoxia or normoxia. Bars represent the means ± standard deviations (triplicates from three independent experiments, normalized to protein concentrations). Statistical differences were calculated as: wild-type normoxia vs HIF-1-null normoxia; wild-type hypoxia vs HIF-1-null hypoxia; wild-type normoxia vs wild-type hypoxia. **P0.01.

View larger version (12K): [in a new window]   Fig. 5. Quantification of extracellular matrix expression in wild-type and HIF-1-null chondrocytes. Epiphyseal chondrocytes were cultured under either 0.5% or 20% oxygen for 22 hours. (A) Analysis of aggrecan gene expression: Bars represent -fold changes of aggrecan mRNA levels in HIF-1-null cells compared with wild-type levels. (B) Alcian-blue staining of extracellular proteoglycans. Bars represent means of alcian-blue absorbance ± standard deviations of wild-type and HIF-1-null cultures exposed to normoxia or hypoxia (duplicates from three independent cultures). Data are given as mean ± standard deviations (triplicates from three independent experiments) and statistical differences were calculated as: wild-type normoxia vs HIF-1-null normoxia; wild-type hypoxia vs HIF-1-null hypoxia; wild-type normoxia vs wild-type hypoxia. **P0.01.

View larger version (12K): [in a new window]   Fig. 6. Analysis of type-II collagen expression in normal and HIF-1-deficient chondrocytes. Epiphyseal chondrocytes were cultured under normoxic or hypoxic conditions for 22 hours. (A) Analysis of type-II collagen gene expression. Bars represent -fold changes of type-II collagen mRNA levels in HIF-1-null compared with wild-type chondrocytes (normoxic expression taken as 1.0). Data are given as mean ± standard deviations (triplicates from three independent experiments). (B) Quantification of type-II collagen protein. Bars represent means ± standard deviations (triplicates from three independent experiments, normalized to total protein content). Statistical differences were calculated as: wild-type normoxia vs HIF-1-null normoxia; wild-type hypoxia vs HIF-1-null hypoxia; wild-type normoxia vs wild-type hypoxia. *P0.05; **P0.01.

View larger version (13K): [in a new window]   Fig. 7. Effect of chemical glycolysis inhibition on type-II collagen expression. Wild-type epiphyseal chondrocytes were incubated with or without 0.2 M 2-deoxyglucose, cultured under normoxic or hypoxic conditions for 22 hours, and type-II collagen gene expression was analysed by quantitative real-time PCR. Bars represent -fold changes of mRNA levels compared with untreated values (normoxic expression taken as 1.0). Data are given as mean ± standard deviations (triplicates from three independent experiments). **P0.01.

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